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RESEARCH ARTICLE
Domesticated cynomolgus monkey embryonicstem cells allow the generation of neonatalinterspecies chimeric pigs
Rui Fu1,2, Dawei Yu1,2, Jilong Ren1,2, Chongyang Li1,2,3, Jing Wang1,2,3, Guihai Feng1,2, Xuepeng Wang1,2,Haifeng Wan1,2, Tianda Li1,2, Libin Wang1,2, Ying Zhang1,2,3, Tang Hai1,2&, Wei Li1,2,3&, Qi Zhou1,2,3&
1 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing100101, China
2 Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China3 University of Chinese Academy of Sciences, Beijing 100049, China& Correspondence: haitang@ioz.ac.cn (T. Hai), liwei@ioz.ac.cn (W. Li), zhouqi@ioz.ac.cn (Q. Zhou)
Received August 6, 2019 Accepted October 26, 2019
ABSTRACT
Blastocyst complementation by pluripotent stem cell(PSC) injection is believed to be the most promisingmethod to generate xenogeneic organs. However, ethi-cal issues prevent the study of human chimeras in thelate embryonic stage of development. Primate embry-onic stem cells (ESCs), which have similar pluripotencyto human ESCs, are a good model for studying inter-species chimerism and organ generation. However,whether primate ESCs can be used in xenogenousgrafts remains unclear. In this study, we evaluated thechimeric ability of cynomolgus monkey (Macaca fasci-cularis) ESCs (cmESCs) in pigs, which are excellenthosts because of their many similarities to humans. Wereport an optimized culture medium that enhanced theanti-apoptotic ability of cmESCs and improved thedevelopment of chimeric embryos, in which domesti-cated cmESCs (D-ESCs) injected into pig blastocystsdifferentiated into cells of all three germ layers. Inaddition, we obtained two neonatal interspecies chi-meras, in which we observed tissue-specific D-ESCdifferentiation. Taken together, the results demonstratethe capability of D-ESCs to integrate and differentiateinto functional cells in a porcine model, with a chimeric
ratio of 0.001–0.0001 in different neonate tissues. Webelieve this work will facilitate future developments inxenogeneic organogenesis, bringing us one step closerto producing tissue-specific functional cells and organsin a large animal model through interspecies blastocystcomplementation.
KEYWORDS embryonic stem cells, blastocystcomplementation, cynomolgus monkey, pig, interspecieschimera, organ reconstruction
INTRODUCTION
Human pluripotent stem cells (hPSCs) have the ability toself-renew and generate a large variety of cell types, andmay be capable of generating human organs in othermammals for future organ transplantation by xenogenesis(Wu et al., 2016). Previous studies have demonstrated thefeasibility of generating an entire organ by xenogenesis,using rodent PSC blastocyst complementation. Hiromitsuet al. used rat PSCs to complement pancreatic and duodenalhomeobox 1 (Pdx1)-deficient murine blastocysts, and gen-erated an entire rat pancreas with normal physiologicalfunction inside a Pdx1-null murine host (Kobayashi et al.,2010). A similar study reconstituted a rat thymus in a nudemouse (Isotani et al., 2011).
However, xenogenesis is more complex in humans thanin mice because hPSCs exist in a “primed” state (Rossant,2015). Primed hPSCs, which show some similarities tomouse epiblast stem cells (EpiSCs) (Nichols and Smith,
Rui Fu, Dawei Yu, Jilong Ren and Chongyang Li have contributedequally to this work.
Electronic supplementary material The online version of thisarticle (https://doi.org/10.1007/s13238-019-00676-8) contains sup-
plementary material, which is available to authorized users.
© The Author(s) 2019
Protein Cell 2020, 11(2):97–107https://doi.org/10.1007/s13238-019-00676-8 Protein&Cell
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2009), can participate in normal mouse development whentransplanted into gastrula-stage embryos. Region-selectivehPSCs in a primed state could also generate post-implan-tation interspecies chimeric embryos after grafting to theposterior epiblast of gastrula-stage mouse embryos (Wuet al., 2015). Although primed hPSCs could not participate innormal mouse development when transplanted into preim-plantation embryos as naïve PSCs (Mascetti and Pedersen,2016), inhibiting apoptosis was shown to enhance the chi-meric ability of primed hPSCs in mice (Huang et al., 2018;Wang et al., 2018b). In addition, Kang et al. showed thatprimed PSCs from cynomolgus monkeys (Macaca fascicu-laris) could generate chimeras using optimal culture condi-tions (Kang et al., 2018). Several recent studies have alsoreported the generation of naive PSCs, which showed higherefficiency in single-cell cloning and genome editing, andcould be incorporated in the host when transplanted intopreimplantation embryos (Gafni et al., 2013; Chen et al.,2015). Moreover, an intermediate primed state, between thenaïve and primed state, was proven to contribute to thegeneration of chimeras by blastocyst injection (Tsukiyamaand Ohinata, 2014), and resulted in better developmentalability of a chimeric pig (Wu et al., 2017).
Presently, mouse models are primarily used as chimerichosts. However, these models are not suitable for transla-tional chimeric research because of the marked differencesin development (i.e., embryo size and gestational period)between rodents and humans. Izpisua et al. pioneered chi-meric research in a large animal model, successfully incor-porating a xenogenous graft of hPSCs into pig blastocystsand confirming hPSC survival in early stage porcineembryos (Wu et al., 2017). However, ethical issues preventthe study of human chimeras in the late stage of embryonicdevelopment, and it is unknown whether human cells cancontribute to the organs of chimeras. Primate PSCs, whichhave similar pluripotency to hPSCs, are a good model forstudying interspecies chimerism and organ generation.
In this study, we used cmESCs to explore the possibilityof interspecies chimerism in the development of lateembryonic stage pigs. By optimizing the medium in which thecmESCs and injected blastocysts were cultured, weobserved an increase in the anti-apoptotic ability of cmESCsand in the extent of chimeric embryo development, resultingin the successful incorporation of xenogenous cmESC graftsinto multiple tissues of the neonatal pigs. This work willenable developments in xenogeneic organogenesis towardsproducing tissue-specific functional cells and organs in largeanimal models via interspecies blastocyst complementation.
RESULTS
Chimeric competency of cmESCs in differentpluripotent states
To systematically evaluate the chimeric competency ofcmESCs in pigs, we prepared cmESCs in three pluripotent
states: primed (P) (Thomson et al., 1998), intermediate(FAC) (Tsukiyama and Ohinata, 2014; Wu et al., 2017), andnaïve (NHSM) (Gafni et al., 2013; Chen et al., 2015). Cells inall three states expressed the pluripotency factor POU class5 homeobox 1 (POU5F1), differentiated into embryoid bod-ies in vitro and teratomas in vivo, which contained tissuesderived from all three embryonic germ layers, and alsoshowed the normal the karyotypes (Fig. S1A–D). To deter-mine whether the cmESCs could be engrafted into the por-cine inner cell mass (ICM), we injected 10 GFP-labeledcmESCs from each pluripotent state into porcine partheno-genetic (PA) blastocysts, followed by 48 h of culture in vitro,and then evaluated their chimeric contributions (Fig. 1A).Three criteria were used to evaluate the chimeric contribu-tions of the cmESCs in porcine blastocysts: survival ofcmESCs in the embryonic environment (the percentage ofGFP-positive embryos divided by the number of embryos),the ability and efficiency of cmESCs to incorporate into totalblastocysts (the number of anti-human nuclear antigen(hNA) antibody-positive cells divided by the number of cells),and their ability to incorporate into the ICM (the number ofanti-hNA antibody-positive cells among the Nanog home-obox (NANOG)-positive cells). There were no significantdifferences in cmESC survival between the different states(Fig. S1E and S1F). However, the average numbers ofcmESCs integrated into each blastocyst in the intermediatestate (27.2 ± 2.107, P < 0.05) and naïve state (30.1 ± 3.585,P < 0.05) were higher than in the primed state (16.4 ± 2.504).Furthermore, the average number of cmESCs integrated intothe ICM in the intermediate state (3.9 ± 0.458) was higherthan in the primed states (1.5 ± 0.401, P < 0.01) (Fig. 1B–D).These results indicated that in vitro, cmESCs in the inter-mediate pluripotent state had better survival and ICM inte-gration ability in pigs compared with the other cell types.
We next investigated whether cmESCs in the differentstates contributed to neonatal porcine development in vivo.In total, 1,203 blastocysts derived from chimeric embryoswere transferred to surrogate sows after 24 h of in vitroculture. Surprisingly, no natal chimeras survived, regardlessof the culture system (Table S1). We suspected that 24 h ofin vitro culture resulted in fatal damage to embryonicdevelopment, especially among the in vitro embryos, whosequality may have been worse than that of embryos fertilizedin vivo, making them more sensitive to external factors.
Domesticated ESC medium positively affectsboth ESCs and embryos during chimeric generation
Since in vitro culture of chimeric blastocysts appeared tohave damaging effects on the embryos, we sought toimprove the chimeric system. Previous studies havedemonstrated that overexpression of anti-apoptotic genessignificantly improves the chimeric ability of human ESCs inmice. We therefore hypothesized that inhibition of apoptosismight enable the cmESCs to form interspecies chimeras
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upon injection into porcine embryos. To test this, we used adoxycycline-inducible system for transient induction of thehuman anti-apoptotic gene BCL2 like 1 (BCL2L1) (Wanget al., 2018b). The survival of cmESCs overexpressingBCL2L1 was improved compared with control cmESCs, andthe average number of BCL2L1-overexpressing cmESCs
integrated into each blastocyst in vitro was higher as well(Fig. S2A–C). However, we still did not obtain any neonatalchimeras from a total of 643 blastocysts transferred intosurrogate sows (Table S1), indicating that other factorsinfluenced interspecies chimera formation.
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Figure 1. Chimeric competency of cmESCs in different pluripotent states. (A) Experimental procedures used to evaluate the
chimeric competency of cmESCs in three pluripotent states by blastocyst injection. (B) Representative images of porcine PA embryos
showing integrated hNApositivity, expressionofNANOG (an ICMmarker), andDAPI staining (amarker of total cells) in cultured chimeric
embryos. White arrows, hNA positive/DAPI cells; yellow arrows, hNA positive/NANOG positive cells; scale bars, 50 µm. (C) Number of
cmESCs in different pluripotent states integrated into the total porcine blastocyst. (D) Number of cmESCs in different pluripotent states
integrated into the porcine ICM. *P < 0.05; **P < 0.01, Student’s t-test. Error bars represent the mean ± SEM (n = 10).
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A comparison of the cell and embryo culture systemsshowed that the pH and osmotic pressure of the cell culturemedium and embryonic medium (EM) differed (data notshown). These differences may have reversed the chimericprocess, leading to in vivo embryonic development failureafter in vitro culture. Thus, we improved the cell culturemedium to better resemble the EM, by mixing FAC medium(FM) with EM, and changing the FM:EM ratio from 3:1 to 1:1(Fig. 2A). We named this domestic medium (DM), and ter-med cmESCs cultured in 1:1 FM:EM domesticated ESCs (D-ESCs), which could be cultured for long periods. Theyexhibited normal ESC morphology (Fig. 2B) and the
karyotypes (Fig. S2D), expressed the pluripotency markersPOU5F1 and SRY-box transcription factor 2 (SOX2; Figs. 2Cand S2E), and in vivo, differentiated into teratomas thatcontained tissues of all three embryonic germ layers(Fig. S2F). Subsequently, we assessed the transcriptomesof primed cmESCs and D-ESCs using RNA sequencing(RNA-seq). The results revealed no significant differences inRNA expression between D-ESCs and cmESCs. Principalcomponent analysis revealed that D-ESCs displayed thesame clusters as cmESCs (Fig. 2D). In addition, naïvemarkers, such as NANOG and PR/SET domain 14(PRDM14), were expressed at higher levels in D-ESCs
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Figure 2. D-ESC derivation. (A) The strategy for generating D-ESCs from cmESCs. (B) Representative bright-field, fluorescence
and merge images of D-ESC colony morphology. Scale bar, 200 µm. (C) Immunofluorescence images of D-ESCs stained with
POU5F1 and SOX2 antibodies. Scale bar, 100 µm. (D) Principal component analysis of gene expression in D-ESCs, cmESCs, and
muscle cells. (E) Heat map of pluripotent gene expression in D-ESCs, cmESCs, and muscle cells. (F) Representative fluorescence
images of TUNEL-stained porcine PA embryos 0 and 24 h after ESC injection. Scale bars, 50 µm. (G) Proportion of TUNEL-positive/
GFP-positive cells 0 and 24 h after ESC injection. *P < 0.05, Student’s t-test. Error bars represent the mean ± SEM (n = 6).
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compared to cmESCs (Fig. 2E). To examine the effects ofthe improved system on chimerism, we first evaluated theanti-apoptotic ability of D-ESCs, and observed enhancedanti-apoptotic ability during chimera development after the24-h co-culture phase compared with that of cmESCs cul-tured in FM (Fig. 2F–G). We also evaluated the effects of DMon embryo development. The results revealed similar blas-tocyst and hatching rates in DM and EM, which were higherthan in FM (Table 1). These results suggest that DM isfavorable for both ESCs and embryos during chimeradevelopment.
D-ESCs can generate interspecies chimeric embryos
Next, we investigated the potential contribution of D-ESCs topost-implantation development following transfer to surro-gate sows. The embryo manipulation procedures performedare shown in Fig. 3A. In brief, porcine embryos derivedthrough in vitro fertilization (IVF) or nuclear transfer (NT)were cultured in vitro to the blastocyst stage. Then, 10–15D-ESCs were injected into each blastocyst, and embryoswere collected 25–30 days later for further analysis. Of 4,359blastocysts transplanted, 59 embryos were obtained, ofwhich three were chimeric. These chimeric embryos col-lected between 25–30 days were verified by a sensitivegenomic polymerase chain reaction (PCR) assay usingmonkey-specific sequence primers (Fig. 3B). Compared towild-type (WT) embryos, obvious green fluorescent protein(GFP) expression was observed in the fetus 5 (F5) sample.We verified the GFP-positivity of F5 by immunofluorescence(IF) analysis (Fig. 3C). To determine how D-ESCs wereinvolved in germ layer differentiation, we costained for GFPand various lineage markers. Subsets of GFP-positive cellsexpressed the endoderm marker forkhead box A2 (FOXA2),mesoderm marker T-box transcription factor 6 (TBX6), andectoderm marker SRY-box transcription factor 1 (SOX1),suggesting that the D-ESCs could differentiate into all threegerm layers (Fig. 3D).
D-ESCs can generate interspecies chimeric neonatalpigs
To evaluate long-term integration, three pregnancies werecontinued until birth. One of these was spontaneouslyaborted, in two other pregnancies, 10 pups were born but all
died within 1 week. Multiple tissues and organs were col-lected from neonatal pigs and analyzed for the presence ofcynomolgus monkey cells. Two of the pups were chimeras,confirmed by PCR using a monkey-specific sequence primer(Fig. S3A). GFP-positive cynomolgus monkey cells con-tributed to multiple tissues, including the heart, liver, spleen,lung, and skin, but not to others, including the testis andovaries, due to low chimerism in the postnatal pigs (Figs. 4Aand S3B). To further confirm the fates of the D-ESCs in theneonatal pigs, we co-stained for GFP and tissue-specificmarkers. Subsets of GFP-positive cells expressed liver andkidney tissue-specific markers, suggesting that these mon-key cells were not in a pluripotent state but had differentiatedinto hepatocyte nuclear factor 4 alpha (HNF4A)-positive livercells and spalt like transcription factor 1 (SALL1)-positivekidney cells, respectively (Fig. 4B). Through mitochondrialDNA (mtDNA) detection, we found that the chimeric ratio indifferent tissues ranged from 0.001–0.0001 (Fig. 4C). Alldata regarding chimeric development with IVF embryosin vivo are shown in Table 2. Taken together, these resultsdemonstrated that D-ESCs contributed to all three germlayers and various tissues in the embryonic and neonatalphases, indicating successful interspecies chimerismbetween cynomolgus monkeys and pigs.
DISCUSSION
In this study, we injected D-ESCs into porcine blastocysts toobtain neonatal interspecies chimeras. The D-ESCs differ-entiated into all three germ layers in the pig fetuses, con-sistent with a previous study reporting human/pig chimeras(Wu et al., 2017). The human/pig fetuses survived only 28day, and it was not possible to observe whether the chimerichuman ESCs could develop into mature functional cells inpigs for ethical issues. In this study, we confirmed thatmonkey/pig chimeras can form functional hepatocytes andrenal cells in neonatal pigs. These functional cells could beisolated for further research and future clinical application.
Interspecies chimerism has been most successful inrodents, and these studies typically achieve a high propor-tion of chimerism and generate xenogeneic organs(Kobayashi et al., 2010; Isotani et al., 2011). The pluripo-tency levels of rodent PSCs are the highest known, as theyare genuine naïve cells, and they can produce live offspringby tetraploid complementation, which is the most stringent
Table 1. Developmental information of embryo cultured in EM, FM and DM
Groups No. oocytes No. blastocysts (%) No. hatching blastocysts (%)
EM 164 33 (22.10 ± 9.35)a 7 (4.72 ± 4.37)a
FM 226 11 (5.32 ± 3.04)b 1 (0.37 ± 0.64)b
DM 189 41 (22.83 ± 5.37)c 6 (3.11 ± 0.40)c
For statistical comparison, Student’s t-test analysis of variance was employed.a FM versus EM (P < 0.05); bDM versus FM (P < 0.05); cDM versus EM (P > 0.05).
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test of pluripotency (Zhao et al., 2009; Li et al., 2017). Recentstudies on xenogeneic chimerism in mice and rats showedthat the pluripotency of rat ESCs affected kidney organreconstruction in mice (Goto et al., 2019), demonstrating that
the pluripotency of the donor cells influenced xenogeneicchimerism. Primate ESCs exist in a different phase ofpluripotency than rodent ESCs, as they are primed ratherthan naïve (Brons et al., 2007; Nichols and Smith, 2009;
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Figure 3. Generation of post-implantation chimeric embryos. (A) Schematic of the generation and analyses of post-implantation
porcine embryos derived from D-ESC injection into blastocysts. (B) Representative gel images of genomic PCR analyses of D25–D30 porcine embryos using the cynomolgus monkey-specific primers CSF and MIT. A pig-specific marker, SOX2, was used as a
loading control. F1–19, fetus samples; P, pig; CM, cmESCs; Mix, pig/cmESCs (1:1); NC, negative control without genomic DNA; red
asterisk, positive chimeric sample. (C) Representative bright-field, fluorescence, and immunofluorescence images of GFP-labeled
D-ESC derivatives in wild-type (WT) and chimeric (F-5) porcine embryos. Scale bar, 2 mm. (D) Representative immunofluorescence
images showing integrated GFP-positive cynomolgus monkey cells and co-expressed lineage markers, including the endoderm
marker FOXA2, mesoderm marker TBX6, and ectoderm marker SOX1. White arrows, lineage marker positive cells; yellow arrows,
cell positive for both GFP and lineage markers. Scale bar, 50 µm.
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Figure 4. Chimeric neonatal pigs generated from D-ESCs. (A) Representative immunofluorescence images of GFP-labeled
D-ESCs in the heart, liver, spleen, lung, skin, and uterus of a chimeric neonatal pig. Scale bars, 100 µm. (B) Representative
immunofluorescence images showing integrated GFP-positive cynomolgus monkey cells and co-expressed organ markers, including
the liver marker HNF4A and the kidney marker SALL1. Yellow arrows, cells positive for both GFP and organ markers. Scale bar,
50 µm. (C) Representative quantitative genomic PCR analysis of cynomolgus monkey mtDNA in the tissues of chimeric neonatal pigs
(No. 1 and No. 4) derived from blastocyst injection with D-ESCs. A series of cynomolgus monkey-pig cell dilutions (1:10–1:100,000)were run in parallel to estimate the degree of monkey cell integration (orange bars). Red bar, monkey mtDNA control; green bars,
tissue of interspecies chimeras. The dashed line indicates the monkey mtDNA detection level and is equivalent to one monkey cell
per 10,000 porcine cells.
Table 2. Developmental and positive efficiencies of D-ESCs after chimeric operation with IVF embryos in vivo.
ESC lines Sex State Stage ofinjection
No. embryotransferred
No. recipient No. development in vivo
No. D25–30embryos GFP+/total
No. full-termGFP+/total
D-ESC-1 Male Intermediate Blastocyst 1,620 12 2/33 2/7
D-ESC-2 Male Intermediate Blastocyst 1,318 8 1/7 0/3
D-ESC-3 Male Intermediate Blastocyst 1,421 9 0/19 0/0
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Boroviak et al., 2014). The ratio of cmESCs in allogenicchimeras is only 0.1%–4.5% (Chen et al., 2015; Kang et al.,2018), which is significantly lower than with rodents. How-ever, in previous reports, naïve hPSCs have lacked thecompetency to integrate into the mouse embryo (Gafni et al.,2013; Theunissen et al., 2014). In our study, chimeras werenot obtained when the PSCs were transferred into naïvestate or with overexpression of an anti-apoptotic gene. Inagreement with Wu et al., we found that the PSCs cultured inthe FAC condition showed a better chimeric contributionin vitro, and were more likely to generate normal individualsin the chimeric processes (Wu et al., 2017). Overall inter-species chimerism is complex, and in addition to thepluripotency of donor cells, its success involves severalother aspects that facilitate interactions between the trans-planted cells and recipient embryos. In our domesticationsystem, the cell and embryo culture media were mixed in anoptimal ratio, increasing the anti-apoptotic ability of cmESCsand improving the development of pig embryos, and allowingthe successful generation of chimeric animals. Improvementof the culture conditions also played an important role in aprevious homozygous chimera study (Kang et al., 2018).The proportion of chimerism in our study was 10–100 timeshigher than in the previously reported human/pig chimera(Wu et al., 2017). Therefore, optimizing the chimeric systemis an effective method to increase the chimerism ratio.
Interspecies chimerism still has a long way to go beforeclinical application is possible. One obstacle is the need toincrease the efficiency of homozygous chimerism byimproving the pluripotency of cmESCs. There have beenmany studies addressing the acquisition of pluripotent andtotipotent stem cells (Yang et al., 2017; Wang et al., 2018a).Second, information is needed regarding the molecularmechanisms underpinning the evolutionary differences thataffect the efficiency of xenogeneic chimera development.Previous studies have reported successful chimeras con-taining human tissue cells, demonstrated by liver (Azumaet al., 2007), neural stem cell (Cohen et al., 2016), andhematopoietic immune system (Goyama et al., 2015)humanization, indicating that there is no obstacle to theremodeling of xenogeneic organs in human/mouse andhuman/pig chimeras. Here, we have used monkey cells toexplore the potential of reconstructing chimeric humanorgans in a large animal model. We believe this work willfacilitate the development of xenogeneic organogenesis byproviding a better understanding of the processes of xeno-geneic recognition, fate determination, and the proliferationand differentiation of primate stem cells during porcinedevelopment. The findings could pave the way towardovercoming the obstacles in the re-engineering of hetero-geneous organs and achieve the ultimate goal of humanorgan reconstruction in a large animal.
MATERIAL AND METHODS
Ethical approval
All animal experiments were performed in compliance with the
guidelines of the Institute of Zoology, Chinese Academy of Sciences.
The cross-species chimeric experiments were reviewed and
approved by the ethics committee of the Institute of Zoology, Chi-
nese Academy of Sciences.
Cell culture
The cmESCs were a gift from Prof. Li, which were established as
described previously (Chen et al., 2015). In brief, primed cmESCs
were digested into clusters using ethylenediaminetetraacetic acid
(EDTA), reseeded on feeder cells in primed medium (PM), and
infected with simian immunodeficiency virus, harboring the
sequence encoding EGFP driven by a cytomegalovirus (CMV)-en-
hanced chicken β-actin (CAG) promoter (Niu et al., 2010). After two
passages, cmESCs sorted by fluorescence-activated cell sorting
(FCAS) were detached using Accutase (Gibco, A11105-1) to obtain
single cells, which were transferred to NHSM and FAC medium to
generate naïve and intermediate cmESCs, respectively. Dome-
shaped colonies were selected and transferred onto fresh feeder
cells for further amplification. The D-ESCs were derived from
cmESCs cultured in FM by mixing the FM with EM at a 1:1 ratio.
Table S2 provides details about the culture media used.
Pluripotency analysis in an in vivo teratoma assay
To induce teratoma formation, 1 × 106 ESCs were subcutaneously
injected into mice with severe combined immune deficiency. Ter-
atomas were collected after approximately 4 week and fixed in 4%
paraformaldehyde for paraffin embedding and hematoxylin and
eosin staining according to standard procedures.
Embryo micromanipulation and chimera assays
Procedures for porcine oocyte collection, in vitro maturation, IVF, PA
production, somatic cell nuclear transfer (SCNT), and embryo cul-
ture were conducted as previously reported (Whitworth et al., 2014;
Yuan et al., 2017). In brief, porcine ovaries were collected from a
local slaughterhouse and transported to the laboratory within 1 h in
normal saline at 37 °C. Oocytes were collected by aspiration and
cultured for 42–44 h in vitro. Cumulus cells were then removed using
0.1% hyaluronidase, and matured oocytes at the MII stage were
selected for IVF, PA, and SCNT. The embryos were cultured in
porcine zygote medium in 5% carbon dioxide at 38.5 °C. In the
in vitro experiment, 10 GFP-labeled ESCs were injected into porcine
PA blastocysts collected 5 day after activation, followed by 48 h of
culture. In the first 4 h, the injected embryos were cultured in ESC
medium and then transferred to mixed ESC medium and EM at a 1:1
ratio for another 20 h, followed by culture in EM for the final 24 h.
Porcine PA blastocysts were then collected for immunofluorescence
analysis. In the in vivo experiment, 10–15 GFP-labeled ESCs were
injected into IVF or NT blastocysts. After 24 h of in vitro culture, the
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injected embryos were transferred into recipient surrogates 5 day
after estrus onset. Pregnancy was diagnosed by transabdominal
ultrasonography using B-ultrasound (Model LX8000, Kangkaijie
Instruments, China) 25–30 day later and confirmed every 2 week.
Piglets were delivered naturally 114–117 day later.
Immunofluorescence
The samples of cells, embryos (25–30 day), and tissue from
neonatal pigs were fixed for 30 min in 4% paraformaldehyde in
phosphate-buffered saline (PBS; 0.01 mol/L, pH 7.4) at room tem-
perature (RT) and permeabilized with 0.5% Triton X-100 in PBS for
30 min at RT. The samples were then blocked in 1% bovine serum
albumin in PBS for 1 h at RT after three 5 min washes in washing
solution (0.1% Tween-20, 0.01% Triton X-100 in PBS), followed by
incubation with primary antibodies (Table S3) overnight at 4 °C. After
three 5 min washes in washing solution, cells or embryos were
incubated with Alexa Fluor series fluorescent tag-conjugated sec-
ondary antibodies diluted in washing solution for 1 h at RT. After
three washes with washing solution, nuclei were stained with 4’,6-
diamidino-2-phenylindole (DAPI; 10 mg/mL in PBS) for 10 min.
Immediately after three washes with washing solution, cells or
embryos in microdroplets in a dish were imaged using a laser
scanning inverted confocal microscope (LSM 780, Zeiss).
Genomic PCR
Genomic PCR was performed to detect monkey cells in the porcine
fetuses and tissues from neonatal pigs. The genomic DNA of pig
fetuses was extracted from the formalin-fixed, paraffin-embedded
(FFPE) slices using TIANamp Micro DNA Kit (TIANGEN, DP316).
The genomic DNA of tissue from neonatal pigs was extracted using
TIANamp Genomic DNA Kit (TIANGEN, DP304). PCR was per-
formed using PrimeSTAR GXL DNA polymerase (Takara, R051A)
followed by Sanger sequencing. Primer sequences used for PCR
are provided in Table S4.
Quantitative PCR (qPCR) analysis of monkey mtDNA
Analysis of monkey mtDNA was performed using SYBR Green Real-
time PCR Master Mix (Takara, RR430A) on an Agilent Mx3005P
qPCR system. Total DNA was isolated from porcine fetuses and
neonatal porcine tissues. A monkey mtDNA control and a series of
monkey-pig cell dilutions were run in parallel to estimate the degree
of monkey cell contribution in interspecies chimeras. For normal-
ization, an identical set of reactions was prepared using primers
specific for an ultra-conserved non-coding element. Primer
sequences used for genomic qPCR are listed in Table S4.
RNA-seq library preparation and data processing
The cmESCs were cultured in PM, FM, and DM. Porcine PA blas-
tocysts were collected 5 day after activation and cultured in PM, FM,
and DM for 48 h. The cmESCs and porcine PA blastocysts were
then collected for RNA-seq analysis. Total RNA was extracted from
cultured cells using TRIzol reagent (Invitrogen, 15596026). For
RNA-seq library construction, PolyA+ tailed RNA purification was
performed for each sample using an E.Z.N.A. MicroElute RNA
Clean-Up Kit (Omega Bio-tek). Sequencing was performed on an
Illumina HiSeq 4000 sequencer with 150-bp paired-end sequencing
reactions. RNA-seq data on monkey muscle samples downloaded
from the GEO database (GSE102830) (Zhang et al., 2018) were
used as a control. All RNA-seq reads were mapped to the M. fasci-
cularis genome assembly (macFas5) using HISAT2 software (Kim
et al., 2015), using annotated gene structures as templates and
default parameters. Reads with unique genome locations were
reserved for gene expression calculations in Cufflinks (version 2.0.2)
using the option “–GTF”. Heat maps and cluster analysis were
produced using the heatmap.2 and hcluster functions of R, respec-
tively. The accession number for the RNA-seq expression values
reported in this study is GSE.
TUNEL assay
TUNEL assays were performed as previously described (Jachowicz
et al., 2017). Embryos were collected 0 or 24 h after ESC injection
and permeabilized in extraction buffer (50 mmol/L NaCl, 3 mmol/L
MgCl2, 0.5% Triton X-100, and 300 mmol/L sucrose in 25 mmol/L
HEPES, pH 7.4) for 5 min on ice. Next, the embryos were washed
twice in extraction buffer without Triton X-100 and incubated with 1
U/mL of DNase I (Ambion, AM2222) in the same buffer for 5 min at
37 °C. After fixation, TUNEL assays were performed using a Click-iT
TUNEL Alexa Fluor Imaging Assay Kit (Life Technologies) according
to the manufacturer’s instructions. Immunofluorescence was per-
formed as described above.
Statistical analysis
Statistical analyses were performed using GraphPad Prism Soft-
ware (GraphPad Prism 7.00). Each experiment included at least
three independent biological replicates. For statistical comparison,
Student’s t-test and one-way analysis of variance were employed.
All data are presented as the mean ± standard error of the mean
(SEM). *P < 0.05; **P < 0.01. Statistical parameters for specific
experiments, including statistical analysis, statistical significance,
and n values, are reported in the figure legends.
ACKNOWLEDGEMENTS
We thank Dr. Tianqing Li for the gift of cmESCs. We also thank
Shiwen Li, Xili Zhu, Qing Meng, and Xia Yang from the Institute of
Zoology, Chinese Academy of Sciences, as well as Junfeng Hao
from the Institute of Biophysics, Chinese Academy of Sciences for
technical assistance. This work was supported by the Strategic
Priority Research Program of the Chinese Academy of Sciences
(XDA16030400 to W.L.), the National Key Research and Develop-
ment Program (2016YFA0100202 and 2017YFA0104401 to T.H.,
2018YFA0109701 to R.F.), the National Natural Science Foundation
of China (31621004 to Q.Z. and W.L., 31571533 to H.W., and
31701286 to G.F.), and the Key Research Projects of the Frontier
Science of the Chinese Academy of Sciences (QYZDY-SSW-
SMC002 to Q.Z.).
ABBREVIATIONS
BCL2L1, BCL2 like 1; cmESCs, cynomolgus monkey ESCs; D-
ESCs, domesticated cmESCs; DM, domestic medium; EM,
Chimeric pigs made with monkey embryonic stem cells RESEARCH ARTICLE
© The Author(s) 2019 105
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&Cell
embryonic medium; EpiSCs, epiblast stem cells; ESCs, embryonic
stem cells; FM, FAC medium; FOXA2, forkhead box A2; HNF4A,
hepatocyte nuclear factor 4 alpha; ICM, inner cell mass; IVF, in vitro
fertilization; NT, nuclear transfer; Pdx1, pancreatic and duodenal
homeobox 1; POU5F1, POU class 5 homeobox 1; PRDM14, PR/
SET domain 14; PSCs, pluripotent stem cells; SALL1, spalt like
transcription factor 1; SOX1, SRY-box transcription factor 1; SOX2,
SRY-box transcription factor 2; TBX6, T-box transcription factor 6.
COMPLIANCE WITH ETHICS GUIDELINES
Rui Fu, Dawei Yu, Jilong Ren, Chongyang Li, Jing Wang, Guihai
Feng, Xuepeng Wang, Haifeng Wan, Tianda Li, Libin Wang, Ying
Zhang, Tang Hai, Wei Li, and Qi Zhou declare that they have no
conflict of interest. All institutional and national guidelines for the
care and use of laboratory animals were followed.
OPEN ACCESS
This article is distributed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to
the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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